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Abstract:

The invention relates to a method for screening for the effects of
non-genotoxic carcinogens in an animal model. The invention also relates
to animal models that are suitable for use in such a method, and cell
lines derived from these animals for in vitro screening purposes. More
specifically, the invention relates to a transgenic rodent animal which
has been humanised for the nuclear transcription factors CAR, PXR and
PPARα, and in which the endogenous equivalent genes have been
rendered inoperable.

Claims:

1. A method for screening a non-genotoxic carcinogen for safety in
humans, the method comprising exposing a preparation of cells to the
non-genotoxic carcinogen and monitoring for a physiological effect,
wherein the animal is humanised for at least two nuclear transcription
factors selected from the group consisting of PXR, CAR, PPARα and
AHR and wherein the endogenous equivalent genes in the animal have been
rendered inoperable.

2. A method according to claim 1, wherein the animal is humanised for at
least PXR and CAR; PXR and PPARα; CAR and PPARα; PXR and AHR;
PPARα and AHR; or CAR and AHR.

3. A method according to claim 1, wherein said animal is a transgenic
non-human animal which has been humanised for the nuclear transcription
factors CAR, PXR and PPARα, and in which the endogenous equivalent
genes have been rendered inoperable.

4. A method according to claim 3, wherein said animal has been
additionally humanised for the AHR receptor, and in which the endogenous
equivalent gene has been rendered inoperable.

5. A method according to claim 1, wherein said animal is a transgenic
non-human animal which has been humanised for the nuclear transcription
factors CAR, PXR and AHR, and in which the endogenous equivalent genes
have been rendered inoperable.

6. A method according to claim 1, wherein all endogenous equivalent genes
of said animal have been rendered inoperable in all tissues.

7. A method according to claim 1 in which the expression level of the
genes rendered inoperable in said animal is less than 10% of the wild
type expression level.

8. A method according to claim 1, wherein the human transcription factor
gene sequences are inserted at the point in the host animal chromosome
where the endogenous equivalent target genes naturally occur.

9. A method according to claim 1, wherein the human transcription factor
genes are inserted at the point in the host animal chromosome where the
endogenous equivalent target genes naturally occur, replacing the
endogenous equivalent target genes in the host chromosomes.

10. A method according to claim 1, wherein transcription of said human
transcription factor genes in said animal is under the control of one or
more endogenous regulatory sequence(s) of the host animal.

11. A method according to claim 1, wherein transcription of said human
replacement gene sequence in said animal is under the control of the
endogenous human regulatory sequence(s).

12. A method according to claim 1, wherein said animal has additionally
been humanised for one, two or all three of CYP3A4, CYP2C9, and CYP2D6.

13. A method according to claim 1, wherein said animal has additionally
been humanised for MDR and/or MRP.

14. A method according to claim 1, wherein said animal has additionally
been humanised for UGT1A.

15. A method according to claim 1, wherein said animal is a rodent, more
preferably, a mouse.

16. A method according to claim 1, used for efficacy screening, PK/PD
modelling or drug safety testing.

17. A method according to claim 1, wherein the non-genotoxic carcinogen
is a ligand for at least one of PXR, CAR or PPARα.

18. A method according to claim 1, wherein said physiological effect is
metabolism of the non-genotoxic carcinogen.

19. A method according to claim 1, wherein said physiological effect is
hepatomegaly, P450 induction or hepatocellular proliferation.

20. A method for screening a non-genotoxic carcinogen for safety in
humans, the method comprising exposing a preparation of cells to the
non-genotoxic carcinogen in vitro and monitoring for a physiological
effect, wherein the cell is derived from an animal humanised for at least
two nuclear transcription factors selected from the group consisting of
PXR, CAR, PPARα and AHR and wherein the endogenous equivalent genes
in the animal have been rendered inoperable.

21. A transgenic non-human animal which has been humanised for the
nuclear transcription factors CAR, PXR and PPARα, and in which the
endogenous equivalent genes have been rendered inoperable.

22. A transgenic non-human animal which has been humanised for the
nuclear transcription factors CAR, PXR and AHR, and in which the
endogenous equivalent genes have been rendered inoperable.

23. An animal according to claim 21, which has been additionally
humanised for the AHR receptor, and in which the endogenous equivalent
gene has been rendered inoperable.

24. An animal according to claim 21, in which all endogenous equivalent
genes have been rendered inoperable in all tissues.

25. An animal according to claim 21, in which the expression level of the
genes rendered inoperable is less than 10% of the wild type expression
level.

26. An animal according to claim 21, wherein the human transcription
factor gene sequences are inserted at the point in the host animal
chromosome where the endogenous equivalent target genes naturally occur.

27. An animal according to claim 21, wherein the insertion of the human
transcription factor genes at the point in the host animal chromosome
where the endogenous equivalent target genes naturally occur replaces the
endogenous equivalent target genes in the host chromosomes.

28. An animal according to claim 21, wherein transcription of said human
transcription factor genes is under the control of one or more endogenous
regulatory sequence(s) of the host animal.

29. An animal according to claim 21, wherein transcription of said human
replacement gene sequence is under the control of the endogenous human
regulatory sequence(s).

30. An animal according to claim 21, which has additionally been
humanised for one, two or all three of CYP3A4, CYP2C9, and CYP2D6.

31. An animal according to claim 21, which has additionally been
humanised for MDR and/or MRP.

32. An animal according to claim 21, which has additionally been
humanised for UGT1A.

33. An animal according to claim 21, which is a rodent, more preferably,
a mouse.

34. A cell isolated from an animal according to claim 21.

35. A cell according to claim 34 which is a stem cell.

36. A cell according to claim 35 which is an embryonic stem cell.

Description:

[0001] The invention relates to a method for screening for the effects of
non-genotoxic carcinogens in an animal model. The invention also relates
to animal models that are suitable for use in such a method, and cell
lines derived from these animals for in vitro screening purposes.

[0002] A significant proportion of therapeutic drug candidates fail to
become marketable drugs because of adverse metabolism or toxicity
discovered during clinical trials. These failures represent a very
significant waste of development expenditure and consequently there is a
need for new technologies that can more reliably, quickly and
economically predict at the pre-clinical development stage the metabolic
and toxicological characteristics of drug candidates in man. At present,
most pre-clinical metabolic and toxicity testing of drug candidates
relies on laboratory animals, human and/or mammalian cell lines and/or
tissues in culture. However, none of these methods is completely reliable
in predicting metabolism or toxicity in a human subject. Metabolic and
toxicological data from animals can differ significantly from that
obtained from a human subject due to species differences in the
biochemical mechanisms involved. In addition, interpretation of data
derived from in vitro human cell cultures or isolated human tissue
studies can be problematic since such systems are not available for all
organs and tissues or they fail to retain the same metabolic
characteristics as they possess in vivo.

[0003] A major factor associated with the assessment of the safety of
drugs and other chemical agents to which we are exposed is their capacity
to induce epigenetic carcinogenesis through the induction of liver
growth. The capacity of agents to act in this manner is currently tested
in laboratory rats or mice; however, it has been demonstrated that these
tests do not necessarily reflect the human situation.

[0004] It is known in the prior art that the metabolism, distribution and
toxicity of most drugs depends on their interactions with four distinct
main classes of proteins, which are phase-1 drug-metabolising enzymes,
such as the cytochromes P450; phase-2 drug-metabolising enzymes, such as
transferases, in particular the glucuronyl transferases, glutathione
transferases, sulphonyl transferases and acetyl transferases; drug
transporter proteins, such as the ATP-binding cassette proteins; and
finally transcription factors, such as the pregnane X receptor (PXR) and
the constitutive androstane receptor (CAR) which regulate the
transcription of genes encoding proteins of the preceding classes, in
particular the cytochromes P450.

[0005] There are a number of reports on mouse lines that have been
humanised for particular transcription factors (see Xie et al, Nature Vol
406, 435-9, 2000; or Zhang et al, Science Vol 298, 422-4, 2002) A
disadvantage of these models is that the PXR or CAR genes themselves are
not regulated as they are in the human by virtue of the transgene being
driven by a heterologous tissue-specific promoter (albumin promoter).
Consequently, over-expression of the heterologous gene can occur, leading
to the result that a normal metabolic pathway is bypassed. Moreover, the
PXR and CAR transgenes are derived from a cDNA rather than a genomic
clone, thus the transgenic non-human animals consequently lack the
sequences necessary correctly to reproduce all the transcriptional and
post-transcriptional regulation of PXR or CAR expression hence their
expression is restricted to the liver and may not be of a physiological
level. In addition these models do not encode for splice variants of the
human gene.

[0006] Our own group has generated a mouse that is double-humanised for
CAR and PXR, against a null background of endogenous expression. As yet,
however, mouse lines that are humanised for more than two of these
receptors have not been generated, particularly where the endogenous
nuclear receptors have been deleted. To our knowledge, no one has yet
suggested the utility of any of these mice to elucidate the relevance of
non-genotoxic carcinogens to human safety evaluation and risk assessment,
or suggested that more complex models might be generated along these
lines.

SUMMARY OF THE INVENTION

[0007] According to a first aspect of the invention, there is provided a
transgenic rodent animal which has been humanised for the nuclear
transcription factors CAR, PXR and PPARα, and in which the
endogenous equivalent genes have been rendered inoperable. Such an animal
is considered to be of great potential in screening for non-genotoxic
carcinogens.

[0008] A number of transcription factors have been identified which
mediate the hyperplastic and hypertrophic effects of chemical agents,
which generally occur on the liver. The inventors consider that out of
these, of particular importance are the nuclear receptors PPARα,
CAR and PXR in the regulation of the cell cycle and growth by
non-genotoxic carcinogens. This aspect of the invention is thus based on
the creation of a transgenic animal that has been humanised for all three
of these receptors and where the endogenous host animal genes have been
concomitantly rendered inoperable.

[0009] This invention provides in one single animal model a predictive
approach to assessing the potential liabilities of drugs in development
and of other chemical entities, that has many advantages over models
described in the prior art. The generation of animal lines according to
the invention markedly increases our understanding of the factors which
determine drug and chemical responses in man. These models can be applied
in a number of different screening scenarios, including, for example,
efficacy screening, PK/PD modelling and drug and chemical safety testing.

[0010] Carcinogens can be classified as genotoxic or non-genotoxic.
Genotoxins cause irreversible genetic damage or mutations by directly
(either the parent molecule or a metabolite of the parent molecule)
binding to DNA; there is generally no systemic level of such compounds
that is considered safe. In contrast, non-genotoxins do not directly
damage DNA but act in other ways to promote growth. Such toxins are
classified as either cytotoxic or non-cytotoxic (no cell necrosis
caused). Examples of non-genotoxins include hormones and some organic
compounds. For all these compounds, there is a threshold level of
exposure that is acceptable for human contact, without risk.

[0011] Current animal models are far from ideal as screens for
non-genotoxic carcinogens, because the rodent receptors that are
regulated by these compounds exhibit well-defined differences in their
ligand specificity between mice, other rodents and humans.

[0012] Accordingly, a compound can appear toxic in the mouse, when its
equivalent effect in the human would be benign or irrelevant. For
example, recent evidence supports the contention that the ligand binding
domains of the murine and human CAR proteins are divergent relative to
other nuclear hormone receptors, resulting in species-specific
differences in xenobiotic responses (Huang et al., 2004, Molecular
endocrinology 18(10):2402-2408). Results reported in this paper
demonstrate that a single compound can induce opposite xenobiotic
responses via orthologous receptors in rodents and humans. Similar
differences exist in the hamster and guinea pig.

[0013] One example of such a drug is Phenobarbital, which is currently on
the market. When tested in rats and mice, both hyperplastic and
hypertrophic effects are seen within days and liver tumours are evident
after around 2 years. Hyperplasia is defined as a proliferation of cells
within an organ or tissue beyond that which is ordinarily seen.

[0014] Hypertrophy also involves an increase in the size of an organ or
area of the tissue, but involves an increase in the size of cells, with
their number staying the same.] Although epidemiological studies show
that phenobarbital does not cause cancer in humans, certain regulatory
authorities are reluctant to ignore the rodent carcinogenicity data when
assessing the safety of other products where it is too early to
demonstrate their safety by retrospective epidemiology studies. As a
result, there is an understandable reluctance to develop drugs and other
chemicals which show hyperplastic effects in the mouse, since it is not
possible to test accurately for hyperplasia in response to a drug without
actually administering the drug to humans. This is clearly unacceptable
unless the drug is known to be safe.

[0015] Another example is the lipid-lowering fibrate drugs, which function
as agonists for the nuclear receptor peroxisome proliferator-activated
receptor α (PPARα). Sustained activation of PPARα leads
to the development of liver tumours in rats and mice (Cattley et al,
1998, 2004). However, humans appear to be resistant to the induction of
peroxisome proliferation and the development of liver cancer in response
to fibrate drugs.

[0016] A further example is provided by the family of peroxisome
proliferator activated receptors (PPARs), to which various drugs were in
the past developed as hypolipidic agents. The development of these drugs
was stopped, as they were identified in mouse and frequently rat models
to be non-genotoxic (also called epigenetic) carcinogens. It was
initially thought that these differences were due to differences in
expression level. However, it turns out that, for unknown reasons the
human receptor upon ligand binding does not activate the cell
proliferation machinery in the same way as the mouse receptor does.

[0017] In vitro screens often use human cells in an attempt to overcome
the problems with cross-species variation. However, in vitro systems can
only ever incorporate a small part of the drug metabolism landscape, and
do not present a holistic view. Accordingly, the real in vivo effects may
be disguised. For example, consider the frequent situation that arises
when drugs look hazardous in vitro because a toxic by-product is
generated but not in vivo because the drug activates a secondary enzyme
that metabolises away the toxic by-product. It is also true that almost
any compound will interact with a particular target at some level--the
question of importance for drug safety is whether this interaction is
physiologically relevant at the concentrations to which tissues will be
exposed. The limitations inherent in the in vitro scenario make this
solution inappropriate.

[0018] It would be of great utility if it were possible to demonstrate
that a hyperplastic response does not occur in humans in response to drug
exposure. The inventors have concluded that one effective way to generate
a faithful test for safety of non-genotoxic carcinogens is through the
use of rodents that have been humanised for the transcription factors
with which non-genotoxic carcinogens principally interact. At the same
time, it is essential to annul the expression of the equivalent
endogenous rodent transcription factor genes in order to ensure that
interference from non-human metabolic pathways on the functions of
introduced human proteins is significantly reduced.

[0019] The inventors have noted that in general, compounds that are
non-genotoxic carcinogens and cause liver tumours in rodents are PXR, CAR
and PPARα ligands. These cause hyperplasia, by either or both
stimulating cell proliferation and inhibiting apoptosis. Additionally,
they cause hypertrophy, stimulating organelle (eg. smooth endoplasmic
reticulum, peroxisomes) proliferation through the smooth endoplasmic
reticulum, and enzyme induction. The barbiturates induce primarily the
P450 enzyme CYP2B; steroids primarily induce CYP3A, and peroxisome
proliferators primarily induce CYP4A. Some chemicals interact
substantially with multiple receptors and induce multiple cytochromes
P450. FIG. 1 is the inventors' depiction of how drug compounds interact
with nuclear receptors and the ensuing metabolic pathways involved in
drug metabolism, cell cycle regulation and growth.

[0020] Mice that have been individually humanised for CAR, PXR or
PPARα currently exist. Indeed, Cheung et al 2004 monitored various
physiological effects including the increase in liver body weight on
exposure to drug in wild type and PPARα knockout mice, and compare
this response to that seen in humanised mice. The humanised mice showed a
lesser increase in liver body weight and a lack of increased replicative
DNA synthesis (a marker for hyperplasia).

[0021] However, the multiply humanised models of the present invention
provide a significant advantage over the deletion of individual
transcription factors because of the functional redundancy between
members of the same gene families.

[0022] The inventors consider that there are a number of reasons why an
animal that has been humanised for all three receptors simultaneously
will provide a significant improvement over the application of the
current singly humanised models.

[0023] In the first instance, the transcription factors that principally
mediate non-genotoxic carcinogens-regulated hyperplasia are all
humanised, so resolving the problems associated with the differences in
ligand specificity noted above. Therefore, one advantage of a humanised
model is that it negates the issues of ligand specificity. All of
PPARα, CAR, and PXR interact with exogenous ligands that
transactivate gene expression, and thereby mediate pathways that the
inventors consider to be potentially deleterious in the metabolism of
non-genotoxic carcinogens.

[0024] The ratio of protein levels that are generated by a particular drug
are also of significant importance. For example, the action of mouse PXR
stimulates expression of different proteins than the action of human PXR
and at different levels. The levels of a particular drug and its
metabolites depends crucially on which drug metabolising enzymes and
transporters are expressed and so, again, the inventors consider that it
is of utmost importance for human transcription factors to be used rather
than endogenous transcription factors from the test animal.

[0025] The use of the specified human transcription factors is also
important from a toxicological standpoint. For example, PXR is naturally
regulated by bile acids and other physiological compounds and toxic
conditions such as biliary necrosis and biliary cholestasis can result
from exposure to a particular drug. It may therefore be that as a result
of differences between drug metabolism between human and a test animal, a
toxic effect will be noted in that animal that would not be evident in
the human.

[0026] One major advantage of these triple humanised animals on a triple
null background is that there is significant redundancy between these
transcription factors in their response to chemicals, as one chemical
agent may interact with multiple receptors. Therefore, a mouse humanised
for just one of these receptors would not give the correct magnitude of
response.

[0027] Furthermore, there are ways in which cross-talk may occur between
different nuclear receptors that are implicated in the metabolism of
non-genotoxic carcinogens. The first is cross-talk between receptors at a
molecular level. The second is cross-talk at a metabolic interface, for
example through generation of cross-reacting secondary metabolites, or
from changes in drug disposition. Thirdly, the nuclear receptors
themselves can cross-talk and modulate each others' levels of expression
and functions. A particular level of drug may activate genes that
transactivate other genes, so leading to further levels of complication.

[0028] Therefore, only by having a complex panel of humanised receptors
will it give the bona fide response that is anticipated in man. Such
cross-talk might in principle be predicted at some qualitative level, but
because the magnitude of the effects and the extent of any feedback
mechanisms are inherently unpredictable, this undermines the value of any
system that does not incorporate all the necessary elements of the system
at physiologically relevant levels.

[0029] One example where cross-talk between these receptors may be of key
importance in defining the eventual outcome is the fact that CAR, PXR and
PPARα transcription factors, in addition to their activation by
exogenous ligands, are regulated by perturbations in fatty acid
homeostasis. It will be advantageous, therefore, to have mice that are
humanised for all these receptors simultaneously.

[0030] Also, the simultaneous humanisation of animals at all three gene
loci dramatically reduces the number of animals required to establish
clearly whether a chemical agent has the capacity to induce hyperplasia,
as it negates the need for carrying out multiple experiments on
individual humanised animals.

[0031] The inventors have noted that the capacity of promoters to induce
enzyme expression is different in different tissues. This adds
significant weight to the contention that human transcription factors
should be used rather than the endogenous transcription factors from the
host animal. Accordingly, the regulatory sequences of the transcription
factors and the genes that they regulate should mirror the natural
physiological condition as closely as possible.

[0032] Animals according to the invention may be any non-human species,
for example a rodent, for instance a rat, hamster or a guinea pig, or
another species such as a monkey, pig, rabbit, or a canine or feline, or
an ungulate species such as ovine, caprine, equine, bovine, or a
non-mammalian animal species. More preferably, the transgenic non-human
animal or mammal and tissues or cells are derived from a rodent, more
preferably, a mouse.

[0033] Although the use of transgenic animals poses questions of an
ethical nature, the benefit to man from studies of the types described
herein is considered vastly to outweigh any suffering that might be
imposed in the creation and testing of transgenic animals. As will be
evident to those of skill in the art, drug therapies require animal
testing before clinical trials can commence in humans and under current
regulations and with currently available model systems, animal testing
cannot be dispensed with. Any new drug must be tested on at least two
different species of live mammal, one of which must be a large
non-rodent. Experts consider that new classes of drugs now in development
that act in very specific ways in the body may lead to more animals being
used in future years, and to the use of more primates. For example, as
science seeks to tackle the neurological diseases afflicting a `greying
population`, it is considered that we will need a steady supply of
monkeys on which to test the safety and effectiveness of the
next-generation pills. Accordingly, the benefit to man from transgenic
models such as those described herein is not in any limited to mice, or
to rodents generally, but encompasses other mammals including primates.
The specific way in which these novel drugs will work means that primates
may be the only animals suitable for experimentation because their brain
architecture is very similar to our own.

[0034] The invention aims to reduce the extent of attrition in drug
discovery. Whenever a drug fails at a late stage in testing, all of the
animal experiments will in a sense have been wasted. Stopping drugs
failing therefore saves test animals' lives. Therefore, although the
present invention relates to transgenic animals, the use of such animals
should reduce the number of animals that must be used in drug testing
programmes.

[0035] The regulatory sequences governing expression of the transcription
factor(s) may preferably be either of human origin, or may originate from
the target animal species e.g. the mouse. Regulation of the expression of
introduced human proteins should be retained such that patterns of
expression in the human are reproduced.

[0036] A further advantage of the invention, particularly where the human
gene is introduced into the endogenous gene locus, is that the predicted
pattern of gene expression is retained. Conventionally, most workers in
this field have integrated a target gene into the host genome randomly,
for example using BAC transgenesis. This strategy has the limitation that
it does not simultaneously introduce any enhancer elements that may lie
large distances up- or down-stream of the replacement gene and so
influence its pattern of gene expression. Furthermore, position effects
on the expression of the transgene are frequently observed. As a result,
non-bona fide expression of the introduced factor will result and, as a
consequence, lead to erroneous results in any experiments using that
animal.

[0037] This also represents an advantage of knocking the transcription
factors into the endogenous gene locus where the effects of downstream
enhancers will still be manifest.

[0038] By "endogenous equivalent gene" of the animal is intended to
include any gene or gene cluster that is functionally capable of
replacing the function which is rendered inoperable, i.e. any gene or
genes whose expression product retains the same, similar or identical
function as the human counterpart gene.

[0039] For example, the human transcription factor gene known as PXR
(NR1I2 nuclear receptor subfamily 1, group I, member 2), Entrez GeneID:
8856, has a murine counterpart of the same name whose Entrez GeneID is
18171. The proteins encoded by these genes have an equivalent function in
the organisms from which they are derived. Accordingly, examples include
acknowledged orthologous counterparts in other organisms. The rat
orthologue has Entrez GeneID 84385.

[0040] The human transcription factor referred to herein as CAR is also
known as NR1I3 (nuclear receptor subfamily 1, group I, member 3) and has
Entrez GeneID 9970. The rat gene is known as Nr1i3 and has Entrez GeneID
65035. The mouse gene is also known as Nr1i3 and has Entrez GeneID 12355.

[0042] The model of the invention may preferably also be humanised for the
nuclear receptor AhR. Similarly, the endogenous equivalent gene should be
rendered inoperable. According to one embodiment of this aspect of the
invention, there is therefore provided a transgenic rodent animal which
has been humanised for at least the nuclear transcription factors CAR,
PXR and AhR, and in which the endogenous equivalent genes have been
rendered inoperable. Such an animal is considered to be of great
potential in screening for non-genotoxic carcinogens. Evidence for the
creation of an animal according to this embodiment of the invention is
described herein. Such an animal may also be transgenic for PPARα.

[0043] AhR is a PAS domain containing protein with a different structure
to the three other nuclear transcription factors mentioned above, and
which has limited or no sequence homology with nuclear receptors. There
are big species differences in ligand responsiveness; even within the
mouse there are polymorphisms that lead to a difference in phenotypes.
The AhR protein induces the activation of enzymes that have an activation
potential to convert promutagens into mutagens. For example, humans are
much less sensitive to dioxins as a result of their AhR transcription
factor. As a hard and fast rule, pharmaceutical companies steer well
clear of any compounds that interact with AhR in order to avoid any
apparent toxicity evident when looking for toxicity.

[0044] AhR is also involved in cross-talk, in a similar way to the other
nuclear receptors described above. For example, interactions are evident
between the receptors AhR and NRF2. Ideally, a system should incorporate
both these elements under appropriate conditions.

[0046] HNF 1 and HNF4 are other examples of transcription factors for
which the animals of the present invention may be humanised, and for
which the endogenous gene may be knocked out.

[0047] Generally, examples of human and murine orthologues of nuclear
transcription factors are known to those of skill in the art.

[0048] Generally, the introduced transcription factor gene will share a
degree of homology with the endogenous gene with which it is equivalent.
Preferably, the degree of homology will be greater than 30%, greater than
40%, greater than 50%, greater than 60%, greater than 70%, greater than
80%, greater than 90%, or even greater than 95%.

[0049] The present invention attempts to mirror the in vivo situation by
providing the replacement gene in its entirety where this is possible.
This means that the intron-exon junctions are retained as in the natural
system so that splicing events can happen exactly as in the natural
situation. Where, perhaps because of the length of a gene, it is not
simple to transpose the entire gene into a transgenic system, the
invention seeks to use a combination of cDNA and genomic DNA in its
constructs so that important intron-exon boundaries, where the majority
of splicing events occur, are retained.

[0050] According to the invention, therefore, where it is known that the
majority of splice variants occur as a result of splicing variation
within a particular intron, this intron is preferably incorporated as
genomic DNA in the construct, while less influential intronic sequences
are not retained. This has the result that levels of functional mRNA and
functional protein mirror the levels that are found in vivo in response
to exposure to a particular drug or drug cocktail. This is what is
ideally required for a physiologically-relevant model.

[0051] Accordingly, whilst cDNA sequences may be used, in preference to
these sequences, the invention may use a combination of cDNA and genomic
sequences from the gene that is to be humanised. For example, in the case
of a transgenic animal expressing the human PXR gene, due to the large
size of more than 35 kb of the human PXR gene, the intron-exon structure
between exons 4 and 6 is preferably maintained (see WO2006/064197), since
most splice variants are observed in this genomic region, since it is
located within the ligand-binding domain. This advantageously retains the
sequence where most splice variants are observed and is conveniently
located within the ligand-binding domain. Similarly, for PPARα, the
encoding DNA sequence comprises at least part of intron 5 and intron 6 of
the human PPARα gene (see FIG. 4). Homozygous mice humanised for
PPARα have been created, and are described herein. It is found that
in addition to the wild type predicted coding sequence, two splice
variants (termed SV1 and SV2) are generated. Variant SV1 has been
published and is a human specific alternatively spliced variant (Gervois
et al, 1999). Variant SV2 is a new type of transcript with the addition
of a 4 bp (GTAG) out-of-frame insert at the 3' end of exon 5, resulting
in a premature stop codon. The potential functions of truncated
PPARα protein in humanised mice are not known.

[0052] Complete genomic DNA sequences will preferably be used. For
instance, in the case of a transgenic animal expressing the human CAR
gene, the relatively small size of the human CAR, which comprises roughly
7 kb from exon 2-9, makes it simple to retain the complete genomic
structure in the targeting vector. The construct should preferably retain
the intron-exon structure between exons 2 and 9 (see WO2006/064197). This
advantageously retains the complete genomic structure within the
targeting vector and permits coverage of all splice variants of human
CAR. Preferably, the genomic human CAR sequence is fused to the
translational start site of the mouse CAR gene. The human CAR sequence
then contains all genomic sequences of exons 1-9. The 5' and 3'UTRs may
be human or may be retained from the mouse genome. All other parts of the
coding sequences of the mouse CAR gene can be deleted.

[0053] The inventors consider it to be of utmost importance when screening
for non-genotoxic carcinogens that as many as possible of the endogenous
genes encoding proteins with relevant functions are rendered inoperable
in the test animal. One reason for this is that there is a high degree of
redundancy between drug metabolism genes such as transcription factors of
the type with which the invention is concerned.

[0054] By the term "rendered inoperable" is meant that the genes or gene
functions are annulled or deleted. This term is thus intended to include
silencing or deletion or rendering inactive so that the host animal's
endogenous equivalent gene is unable to express the gene product(s), at
least not to any level that is significant to the drug metabolism
process. For instance, the expression level of an annulled gene may be
less than 20%, preferably less than 10%, more preferably less than 5%,
more preferably less than 2%, even more preferably 1% or less of the wild
type expression level. The expression of a gene rendered inoperable may
preferably be decreased to the point at which it cannot be detected.

[0055] Preferably, the endogenous transcription factor functionality is
deleted in all tissues of the animals of the present invention. It is a
widely-accepted misconception that the liver is the only truly important
tissue for drug metabolism. The reality is far from this; in fact
transcription factor function is expressed across a wide range of tissues
other than the liver; particular examples include the gastro-intestinal
tract and the blood brain barrier. The complete abrogation of function
across all tissues is thus necessary in order that the effects of the
endogenous gene knockouts are manifest.

[0056] The models of the invention thus provide advantages over many other
models where a particular transcription factor system has been
inactivated, for example using conditional knock-out in the liver. In
contrast, in the models of the present invention, deletion of the
transcription factors preferably occurs in all tissues.

[0057] One example of a very good reason for the utility of the animal
models of the present invention is that mice, as conventionally used,
metabolise most drugs far more quickly than humans, almost by a factor of
10, generally because of slower rates of P450 metabolism. Accordingly,
mice that activate such enzymes will perhaps exhibit a 30 minute
half-life where the equivalent drug will have a half-life of some hours
in the human. Of course, this has the effect that the normal pathways of
disposition are masked in the mouse, because there is no opportunity for
these to take effect. Accordingly, deleting the dominant transcription
factors that govern non-genotoxic drug metabolism has the effect of
removing these pathways of drug disposition from contention. Humanisation
of such knockout mice for the equivalent human transcription factors then
allows human drug metabolism pathways to be evaluated without
interference from the endogenous mouse enzymes.

[0058] Examples of methods for rendering endogenous equivalent genes
inoperable in the target animal are set out in WO2006/064197. In brief,
the endogenous host gene(s) may be rendered inoperable by a number of
different means, as will be clear to those of skill in the art. For
example, this may be by complete deletion of the coding sequences of the
genes from the host animal genome. Alternatively, deletion may be
accomplished by mutation of the coding sequence, either by way of
insertion, deletion or substitution of other sequences. For example, one
or more mutations (such as frameshift mutations) may be generated such
that any resulting RNA transcript codes for a non-functional or truncated
protein. In an alternative, an insertion may be made into the chromosomal
sequence to disrupt the amino acid code.

[0059] Similarly, a sequence may be exchanged with the transcription
factor sequence that is being deleted, such as a selection or marker
sequence that can be used as the basis for screening for successful
deletants. One such strategy has been devised by Wallace et al (Cell 128,
197-209 2007), in the context of gene exchange, and this is applicable to
the method of the present invention. This method envisages an exchange of
sequence between mouse chromosome and a BAC or YAC vector, such that two
intermolecular homologous recombination events are required for the
vector-based replacement sequence to replace the endogenous genomic
murine sequence.

[0060] In one preferred system, a mechanism of homologous recombination is
used to exchange a gene for an alternative sequence in which the gene is
not present. Such a method preferably comprises the steps of: a)
incorporating a pair of site-specific recombination sites into the host
animal chromosome by homologous recombination such that the target gene
that is to be replaced is flanked on each side by a recombination site;
and b) effecting recombination between the site-specific recombination
sites such that the target gene is excised from the chromosome, replaced
by a residual site-specific recombination site.

[0061] Methods for performing homologous recombination are known in the
art and exploit regions of homology between exogenously supplied DNA
molecules and the target chromosome to introduce the RT sites. Under this
methodology, 5' and 3' homology arms in the replacement sequence drive
recombination between the replacement sequence and target such that the
gene is deleted. A methodology utilising this strategy is reported in the
applicant's co-pending patent application no. GB0718029.2 filed on 14
Sep. 2007 entitled "Two step cluster deletion and humanisation" and
PCT/GB2008/003084, of the same title. This is perfectly applicable to the
method of the present invention.

[0062] In this arrangement, each of the replacement sequences is designed
such that between the 5' and 3' homology arms lies a selection marker and
at least one recombinase target (RT) site such as loxP, lox5171, FRT or
F3. In this manner, it is possible to select for successful incorporation
of both replacement nucleic acids that would thus flank the gene to be
deleted. One of the flanking sequences can be designed so that a
replacement human transcription factor gene sequence lies outside the RT
site. It is then technically simple to excise the gene by exposure of the
cells to an appropriate site-specific recombinase (SSR) that recognises
the RT sites. This effects total deletion of the host animal
transcription factor gene while at the same time replacing the gene with
the human equivalent. These described recombination steps are preferably
performed in an embryonic stem cell, according to methods well known in
the art.

[0063] A preferred strategy for generation of a transgenic line initially
involves the creation of altered embryonic stem cells. The altered
embryonic stem cell may be subsequently inserted into a blastocyst.
Conventionally, blastocysts are isolated from a female mouse about 3 days
after it has mated. Up to 20 altered embryonic stem cells may be
simultaneously inserted into such a blastocyst, preferably about 16.
Through insertion of altered embryonic stem cells into the blastocyst,
the embryonic stem cell will become incorporated into the developing
early embryo, preferably by its transplantation into a pseudo-pregnant
animal, such as a mouse, which has been induced so as to mirror the
characteristics of a pregnant animal. According to this methodology, the
blastocyst, containing the altered embryonic stem cell, will implant into
the uterine wall of the pseudo-pregnant animal and will continue to
develop within that animal until gestation is. complete. The altered
embryonic stem cell will proliferate and divide so as to populate all
tissues of the developing transgenic animal, including its germ-line.

[0064] In one aspect of the methodology, the created transgenic animal may
be a chimera, containing altered and non-altered cells within each
somatic tissue and within the germ-line.

[0066] Ultimately, two heterozygous animals produced according to the
methodology above may be crossed to produce a transgenic animal that is
homozygous for the human allele of the gene or genes of interest.
Crossing two heterozygous transgenic animals will produce a proportion of
progeny that are homozygous for the deletion.

[0067] In a further embodiment of the invention the transgenic non-human
animal is produced de novo so as to include all of the aforementioned
features, by the methods as hereinafter disclosed.

[0068] In another embodiment of the invention the mouse of the present
invention is produced by crossing. For example, a partial deletant in
which a proportion of the genes of a particular cluster have been deleted
could be crossed with another partial deletant to generate animals which
are deleted for all gene functions within a particular cluster.

[0069] Transgenic mice for human CAR have been created and are described
in the examples included in WO2006/064197 and WO2008/149080. Detailed
investigations of the induction of drug metabolism pathways in CAR
humanised and knock-out mice have been performed. Various different
experimental approaches have confirmed that non-human transgenic animals
that are humanised with respect to CAR, or which do not express any CAR
(knock-out), can readily be obtained using the methods and strategies
described herein.

[0070] Transgenic mice for human PXR have also been created and again are
described in the examples included in WO2006/064197 and WO2008/149080.
Human PXR is found to be expressed in both the liver and GI tract of mice
in the predicted manner at levels equivalent to those of the endogenous
mouse gene. In this way, typical problems faced by conventional
techniques of this type, such as over- or under-expression are avoided.
In this model, the PXR protein has also been shown to be functional as
the mice are responsive to compounds such as rifampicin and dexamethasone
that are known to induce gene expression via this pathway. Strain
differences between wild type and the humanised mice have been
demonstrated. For example, the humanised mice are shown to be more
responsive to compounds such as rifampicin, that are known to be more
active to hPXR. Humanised PXR animals thus demonstrated an altered
sensitivity to rifampicin relative to the wild type.

[0071] Furthermore, there was clearly greater background P450 enzyme
activity as measured by 16-beta-hydroxylation of testosterone and
7-benzyloxyquinoline debenzylation between wild type and humanised PXR
mice.

[0072] Transgenic animals (such as mice) and cells according to the
invention preferably demonstrate the functional properties described
above and in the examples herein. For example, such cells and animals
preferably do not display induction of Cyp2b10 activity in response to
rifampicin. However, such cells and animals do display an induction
effect for Cyp3a11, not only with rifampicin but also for TCPOBOP.

[0073] Mice transgenic for both human PXR and human CAR have also been
created and are described in the examples included herein. Preliminary
studies have been performed on the activity of these transcription
factors in combination, determined by measuring barbiturate-induced
sleeping time. Sleeping time has been known for many years to be directly
proportional to the hepatic cytochrome P450 activity and this activity
can be at least in part ascribed to the P450 levels in the liver
determined by CAR and PXR function. Whereas wild type mice given a
narcotic dose of pentobarbitone slept for 21 minutes, the double
humanised mice for CAR and PXR slept for 34 minutes. These mice therefore
demonstrate a significant difference to their wild type controls
indicating that the double humanised mouse has a marked difference in its
response to drugs relative to the wild type animals.

[0074] Detailed investigations of the induction of drug metabolism
pathways in PXR and CAR double-humanised and double-knock-out mice have
been performed. Various different experimental approaches have confirmed
that non-human transgenic animals that are humanised with respect to both
PXR and CAR, or which do not express any PXR or CAR (double-knock-out),
can readily be obtained using the methods and strategies described
herein.

[0075] Given the results described herein, there is no technical barrier
preventing the generation of animals that are also transgenic for
PPARα. The relevant constructs have already been made and are
described herein. Targeting strategies suitable for knock-in
(humanisation) and knock-out of PPARα and the Ah receptor are
described in more detail herein (see FIGS. 4 and 5). At the time of
writing, Mice homozygous for CAR and PPARα and additionally
heterozygous for PXR have been made and triple homozygous mice will be
available shortly. Additionally, mice homozygous for humanised PXR, CAR
and AhR are already available and described in the examples herein.

[0076] Ultimately, the animal models of the invention may be exploited as
a background for introducing human genes that may substitute the
functions of rodent enzymes, either by integrating these genes directly
into the same chromosomal region (and thus replacement of the endogenous
gene(s)) or through integration at alternative sites. Preferably, human
genes will be integrated into the same chromosomal region, since the
integrity of the chromosome will be retained and thus physiological
patterns of expression and tissue distribution are likely to be similarly
retained.

[0077] Accordingly, in embodiments of the invention relating to the
preparation of cells and animals as previously described, such cells and
animals may be subjected to further transgenesis, in which the
transgenesis is the introduction of an additional gene or genes or
protein-encoding nucleic acid sequence or sequences. The transgenesis may
be transient or stable transfection of a cell or a cell line, an episomal
expression system in a cell or a cell line, or preparation of a
transgenic non-human animal by pronuclear microinjection, through
recombination events in non-embryonic stem (ES) cells, random
transgenesis in non-human embryonic stems (ES) cells or by transfection
of a cell whose nucleus is to be used as a donor nucleus in a nuclear
transfer cloning procedure.

[0078] In particular, it is envisaged that an animal according to the
invention may be humanised for one or more human genes, including drug
transporters, transcription factor or phase I drug metabolism enzymes,
phase II drug metabolism enzymes, and so on.

[0079] For example, such an animal may be humanised for phase-1 drug
metabolising enzyme i.e. a P450 enzyme. Preferred examples of P450
enzymes include one, two, three or more of CYP3A4, CYP3A5, CYP2C9,
CYP2C19, CYP2D6, CYP1A1, CYP1A2, CYP2C8 and CYP2B6. The animal may be
humanised for an entire gene cluster, such as the CYP3A cluster, the
CYP2D cluster and/or the CYP2C cluster.

[0080] An animal according to the invention may also be humanised for a
phase-2 drug-metabolising enzyme. Examples of such enzymes include the
glucuronyl transferases, for instance, the UGT1A gene or gene cluster,
the glutathione transferases, for instance GST (glutathione
S-transferases) (including GST-ml and/or t1 clusters), the sulphonyl
transferases and the acetyl transferases.

[0081] It is also preferred that the endogenous equivalent murine genes
have been annulled, as set out in WO2006/064197. In the case of
drug-metabolising enzyme genes, equivalence between genes can be assessed
by a combination of substrate specificity, mode of regulation (for
example, by transcription factors or exogenous drugs), sequence homology
and tissue distribution. Certain genes have exact equivalents; examples
of such genes are CYP2E1, CYP1A1 and CYP1A2. CYP2B6 and CYP2D are
examples where there is only one gene in the human, but numerous
equivalent genes in the mouse. There are four CYP2C genes in the human,
and numerous equivalent genes in the mouse. In such circumstances,
preferably at least one, more preferably two, three, four, five or more
or even all of the equivalent murine genes are annulled. CYP3A4 is an
example where there is no obvious orthologue in the mouse, but Cyp3a11
could be considered at least one equivalent mouse gene because of its
hepatic expression, mode of regulation and sequence homology.

[0082] An animal according to the invention may also be humanised for a
drug transporter protein, examples of which include the multi-drug
resistance proteins, for instance mdr1 and mdr3 and multi-drug
resistance-associated proteins (MRPs), for example, MRP1 and/or MRP2
and/or MRP6 or from the organic anion transporting polypeptides (OATPs).
It is also preferred that the endogenous equivalent murine genes have
been annulled.

[0083] Another aspect of the invention relates to cells, modified so as to
possess properties according to any one of the above-described aspects of
the invention. Hepatocytes and neuronal cells are preferred cell types
according to the present invention. The cells may be rodent cells, in
particular, mouse cells.

[0084] Cells according to this aspect of the invention may be created from
transgenic mice according to the invention using standard techniques, as
will be clear to the skilled reader, imbued with knowledge of the present
invention. Suitable methods are described in many standard laboratory
manuals, such as Davis et al., Basic Methods in Molecular Biology (1986);
Sambrook Molecular Cloning; A Laboratory Manual, Third Edition (2000);
Ausubel et al., 1991 [supra]; Spector, Goldman & Leinwald, 1998).

[0085] One preferred method of generating such cells is to cross a
humanised mouse, as described above, with SV40 immortalised mouse (for
example, the immorta-mouse; Taconic). Cells may subsequently be isolated
from such animals according to well known techniques in the art. In
contrast to prior art transgenic systems, which used the albumin promoter
that is only active in the liver and thus only able to generate
hepatocytes, cells from transgenic animals generated according to the
present invention may be of a diverse selection of different cell types,
including cells of significant importance to pharmacokinetics analyses,
such as hepatocytes and neuronal cells.

[0086] Stem cells isolated from transgenic animals according to the
invention, with properties as described above are also useful aspects of
the present invention. Such cells may be pluripotent, or partially
differentiated. Stem cells may be adult stem cells or embryonic stem
cells. More generally, stem cells employed may be from a post-embryonic
developmental stage e.g. foetal, neonatal, juvenile, or adult. Stem cells
isolated in this manner may be used to generate specific types of cells
such as hepatocytes and neuronal cells. Such cells also form an aspect of
the present invention.

[0087] Cells or animals produced by the method of the invention can be
used as model systems for determining the metabolism of drugs or other
xenobiotic compounds in other organisms, particularly the human.

[0088] In a still further aspect of the invention, an assay according to
the invention involves a method for screening a non-genotoxic carcinogen
for safety in humans comprising exposing a non-human animal to the
non-genotoxic carcinogen and monitoring for a physiological effect,
wherein the animal is humanised for at least two nuclear transcription
factors selected from the group consisting of PXR, CAR and PPARα,
or the group consisting of PXR, CAR, AhR and PPARα, and wherein the
endogenous equivalent genes have been rendered inoperable. The animal may
be thus be humanised for PXR and CAR; PXR and PPARα; CAR and
PPARα; PXR and AHR; PPARα and AHR; CAR and AHR or three or
all four of these nuclear receptors (for example, PXR, CAR and
PPARα, or PXR, CAR and AhR), in accordance with the terms of the
invention set out in detail above.

[0089] Assays according to the invention may thus be practised on a wider
range of animals than just the animals discussed above. As far as we are
aware, prior to this disclosure, the concept of using mice that are even
doubly humanised for nuclear transcription factors has not been suggested
to screen non-genotoxic carcinogens for safe use in humans. Such
double-humanised models are advantageous over models that only
incorporate a single gene (either PXR or CAR) because many drug
metabolising enzymes or drug transporters possess elements that are
responsive to the binding of both CAR and PXR. Furthermore, the numbers
of PXR-responsive elements often differ from the numbers of
CAR-responsive elements and so regulation by both transcription factors
is generally important. Consequently, models that take account of the
effects of both factors are preferable and more closely mirror the
physiological situation in vivo. Mice transgenic for both human PXR and
human CAR have been created and are described in the examples included
herein. Various different experimental approaches have confirmed that
non-human transgenic animals that are humanised with respect to both PXR
and CAR, or which do not express any PXR or CAR (double-knock-out), can
readily be obtained using the methods and strategies described herein.
Preferably, the mouse models are homozygous for the humanised genes.

[0090] The animals, tissues and cells of the present invention may be used
to determine how a drug compound is metabolised. The generation of animal
lines according to the aspects of the invention described above will
markedly increase our understanding of the factors which determine drug
and chemical responses in man and the relevance of these genes for
chemical toxicity. These models can be applied to efficacy screening,
PK/PD modelling and drug safety testing.

[0091] It is possible to measure a phenotypic change in the animal, such
as a physiological effect. Such a physiological effect may be, for
example, a disease condition (such as biliary necrosis) or a toxic
side-effect. Preferred phenotypic changes include hyperplasia,
hepatomegaly, P450 induction and/or hepatocellular proliferation.

[0092] It is possible to examine the rate of metabolism of a drug
compound. The rate of metabolism may be determined by measuring the
toxicity or activity mediated by the administration of the compound,
measuring the half-life of the compound, or measuring the level of a
transcription factor or drug metabolising enzyme. For example, the rate
of metabolism of the compound may be measured as the rate of formation of
the oxidized product or the formation of a subsequent product generated
from the oxidized intermediate. Alternatively, the rate of metabolism may
be represented as the half-life or rate of disappearance of the initial
compound or as the change in toxicity or activity of the initial compound
or a metabolite generated from the initial compound. The half-life may be
measured by determining the amount of the drug compound present in
samples taken at various time points. The amount of the drug compound may
be quantified using standard methods such as high-performance liquid
chromatography, mass spectrometry, western blot analysis using compound
specific antibodies, or any other appropriate method.

[0093] It is also possible to examine whether under particular
circumstances a drug compound is metabolised to a toxic or carcinogenic
metabolite, for example, by measuring its covalent binding to tissues,
proteins or DNA or by measuring glutathione depletion.

[0094] Preferably, measurements of the type described above are performed
at more than 1, 3, 5, 10 or more time points after administration of the
drug compound.

[0095] Accordingly, further aspects of the invention relate to screening
methods that are provided to determine the effect of a drug compound on
the activity or expression level of a transcription factor, a drug
metabolising enzyme or a drug transporter protein. Such methods involve
administering a drug compound to a transgenic animal according to any one
of the aspects of the invention described above, or a tissue or cell
derived therefrom.

[0096] The screening step may involve measuring the induction of a gene
coding for a transcription factor, a drug metabolising enzyme or a drug
transporter protein. The screening step may involve measuring the level
of expression of a transcription factor, a drug metabolising enzyme or a
drug transporter protein or the duration of such expression. The
screening step may involve measuring the distribution of expression of a
transcription factor, a drug metabolising enzyme or a drug transporter
protein.

[0097] The assay can be performed in the presence and absence of the drug
compound to ascertain differences in distribution, metabolism and
toxicity. The effects of the drug compound in the presence and absence of
a particular gene or genes can be ascertained by evaluating the effects
of the drug compound on different transgenic animals, cells or tissues.

[0098] Thus, in a further aspect the invention provides methods for
investigating xenobiotic metabolism or toxicity as described herein,
comprising administering a drug compound to 2 or more, 3 or more, 4 or
more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or
more of the non-human animals, tissues or cells described herein.
Preferably, such methods further include a step of comparing the
experimental results obtained for different non-human animals, tissues or
cells.

[0099] More than one drug compound may be administered. For example, a
drug compound is considered to activate the CAR transcription factor if
the compound mediates induction of the CAR gene. A CAR receptor inverse
agonist such as clotrimazole can also administered to an animal
expressing the human CAR receptor as a control.

[0100] Assays according to further aspects of the invention may provide a
screening method for determining whether the metabolism of a first drug
compound is modulated by a second drug compound. This method involves
administering the first compound in the presence and absence of the
second compound to a transgenic animal according to any one of the
above-described aspects of the invention, or a tissue or cell derived
therefrom, and monitoring for a phenotypic effect. Alternatively, as
above, the screening step may involve measuring the induction of a gene,
the level, duration or distribution of expression, of a transcription
factor, a drug metabolising enzyme or a drug transporter protein. The
second compound is determined to modulate the metabolism of the first
compound if the second compound effects a change in any one of these
tested factors. For example, a physiological effect may be assayed by
measuring the toxicity or activity mediated by the administration of the
first compound or measuring the half-life of the first drug compound.

[0101] In this manner, assays may be used to facilitate the identification
of analogs of a drug compound that have reduced or undetectable ability
to activate or induce expression of a particular protein, and thus are
expected to have fewer side-effects or a longer half-life in vivo.

[0102] Various aspects and embodiments of the present invention will now
be described in more detail by way of example. It will be appreciated
that modification of detail may be made without departing from the scope
of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0103] FIG. 1 depicts the role of nuclear receptors in metabolic pathways
within the cell.

[0104]FIG. 2 shows the role of PXR/CAR in the metabolism of non-genotoxic
carcinogens.

[0109]FIG. 7 shows the results of an experiment in which humanised,
knockout and wild type mice are exposed to PB treatment. Physiological
effects that are monitored include hepatomegaly, P450 induction and
hepatocellular proliferation.

[0110]FIG. 8 shows the liver/body weight ratios in WT, huPXR/huCAR and
PXRKO/CARKO mice. The values are expressed as Mean±SD (% mean own
control±% SD; n=3. A Student's t-test (2-sided) was performed on the
results; * and ** indicate that the difference is statistically
significant from control mice at p<0.05 and p<0.01, respectively.

[0111]FIG. 9 shows hepatic S-phase Labelling Indices in PB-treated mice.
Osmotic pumps containing BrdU (15 mg/ml/PBS) were implanted into WT,
huPXR/huCAR and PXRKO/CARKO mice prior to PB treatment (80 mg/kg/4
days/IP). Livers sections were labelled using a Brdu antibody (Dako). All
microscopic images were captured at a magnification of 40×. Data
represents random sampling of 10 images per lobes (2) counting
approximately 180,000 cells/animal group, as according to Pat-0013-0014.
Values are expressed as Mean±SD, n=9-10 for control mice, n=8-9 for
PB-treated mice. A Student's t-test (2-sided) was performed on the
results with *** indicating that the difference is statistically
significant from control mice at p<0.01.

[0112]FIG. 10 shows Hepatic apotopic indices in PB-treated mice. Livers
sections were labelled using a TUNEL in situ cell detection kit (Roche).
All microscopic images were captured at a magnification of 40×.
Data represents random sampling of 20 images per lobes (2) counting
approximately 380,000 cells/animal group. Values are expressed as
Mean±SD, n=9-10 for control mice, n=8-9 for PB-treated mice. A
Student's t-test (2-sided) was performed on the results with no
statistical significance found.

[0113] FIG. 11 shows H&E staining on liver sections taken from control and
PB-treated WT, huPXR/huCAR and PXRKO/CARKO mice. Portal vein (P) and
central vein (C) are labelled. A 20× objective lens was used to
capture the images.

[0114] FIG. 12 shows Enzyme activity measurements a) MROD, b) EROD, c) BQ,
d) PROD and e) BROD assays. Each assay was performed on individual liver
microsomes. Values are expressed as Mean±SD (n=9/10). A Student's
t-test (2-sided) was performed on the results; with *, ** and ***
statistically different from own control mice at p<0.05, p<0.01 and
pO.OOl, respectively.

[0115]FIG. 13 shows the effect of PB on hepatic Cyp2b10 and Cyp3a11
protein expression. Liver microsomes (0.3 ug) from each animal were
pooled (n=9/10) for wild type, huPXR/huCAR and PXRKO/CARKO mice and
characterised for Cyp2b10 and Cyp3a11 by immunoblotting using rabbit
polyclonal CH4 (1:2000 dilution) and CH32 antibodies (1:2000 dilution),
respectively (C. Henderson, University of Dundee, UK); +ve, control was
either purified recombinant his-tagged Cyp3a11 membranes (0.1 pmol/u.l)
or purified recombinant his-tagged Cyp2b10 membranes (O.Olpmol/u.l).
Blots were developed using ECL and exposed for 30 secs.

[0116]FIG. 14 shows the PXR/CAR dependant species differences in
hyperplastic response to Phenobarbital

[0117]FIG. 15 shows the PXR/CAR dependant species differences in
hyperplastic response to Chlordane.

[0118]FIG. 16 shows H&E staining on liver sections taken from control and
Chlordane-treated WT, hPXR_old/hCAR and PXRKO/CARKO mice. A 20×
objective lens was used to capture the images. This figure shows
hypertrophy in WT and hPXR_old/hCAR mice but not PXRKO/CARKO upon
exposure to Chlordane.

[0123] FIG. 21 shows the presence of human PPARα in the hPPARα
mouse by RT-PCR. Liver RNA was isolated from a vehicle-treated WT mouse
and a vehicle-treated hPPARα mouse and analysed by RT-PCR using
primer pair PPARα-F and PPARα-R. RT-PCR products from the WT
mouse were loaded into a single well (lane 1), whereas the RT-PCR
products from the hPPARα mouse were loaded in duplicate (lane 2-3).
Two bands were detected at 1.4 kb and 1.2 kb in the hPPARα mouse.
M=molecular weight marker. N.B. the lower bands in each samples are
non-specific.

[0124] FIG. 22 shows wild type, splice variant 1 (SV1) and splice variant
2 (SV2) transcripts of human PPARα detected in the hPPARα
mouse. SV1 is a transcript with deletion of exon 6, resulting in a frame
shift introducing a premature stop codon. SV2 is a transcript using an
alternative intronic 3' splice site resulting in the addition of a 4 bp
(GTAG) out-of-frame insertion at the 3'end of exon 5, also resulting in a
premature stop codon.

[0128] FIG. 26 shows that CAR snRNA is not expressed in the liver or small
intestine of CAR knockout mice.

[0129] FIG. 27 shows methods of monitoring CAR function, and indicates the
species specificity of each method.

[0130] FIG. 28 shows the loss of CAR function in CAR knockout mice,
measured using TCPOBOP, which is most active in mice.

[0131] FIG. 29 shows the loss of CAR function in CAR knockout mice,
measured using CITCO, which is most active in humans.

[0132] FIG. 30 shows the expression of CAR mRNA in CAR humanised mice. The
murine but not the human CAR transcript is expressed in CAR humanised
mice. The full length and all human splice variants of CAR are expressed
in the CAR humanised mice.

[0133] FIG. 31 shows the alternative splicing patterns of human CAR . Two
of the ligand binding domain isoforms demonstrate novel functional
properties. SV3 has differentially transactivated target gene promoters,
and SV2 shows ligand-dependent rather than constitutive interactions with
coactivators. Alternative splicing appears to be of the utmost importance
for the regulation of CAR expression and function.

[0134] FIG. 32 shows murine CAR dependent P450 induction in wildtype and
CAR humanised mice by TCPOBOP, which is most active in mice.

[0135] FIG. 33 shows human CAR dependent P450 induction in wildtype and
CAR humanised mice by TCPOBOP, which is most active in humans.

[0137] FIG. 35 shows the inductive effect of Phenobarbital in a panel of
PXR/CAR knockout and humanised mice. Although Phenobarbital is described
as a PXR activator in vitro, the Phenobarbital-mediated activation of
Cyp3a11 and Cyp2b10 in vivo is predominantly CAR-dependent.

[0139] A description of suitable techniques for the generation of mice
humanised for both PXR and CAR on a null background can be found in
WO2006/064197 (see Examples 3 and 4 and the corresponding Figures).

Example 1

PCR Confirmation in Double Homozygous PXR/CAR Humanised Mice that the
Murine PXR Gene has Been Exchanged for the Human Countepart

[0140] Humanised mice for PXR and CAR ("huPXR/huCAR") were generated using
mice which contained humanised PXR and crossing these into mice which
contained humanised CAR to produce mice containing both humanised PXR and
humanised CAR. The mice are phenotypically normal following visual
inspection. They have been typed using PCR (see FIG. 28 of WO2006/064197)
and are homozygously humanised for PXR and CAR. Examples include mice
designated "42749" and "42752".

[0141] Transcription of PXR and CAR mRNA was quantified by RT-PCR in
huPXR/huCAR mice and compared to the relative levels of corresponding
mRNA expression in wild-type, huPXR and huCAR mice (FIG. 2). It was
thereby confirmed that the huPXR/huCAR mice maintain the levels of human
PXR and human CAR expression observed in mice humanised with respect to
single genes.

[0142] Double-humanised huPXR/huCAR mice, as well as wild-type, huPXR and
huCAR mice were treated with the inducers rifampicin and/or
phenobarbital. Expression of Cyp2b10 and Cyp3a11 in these inducer-treated
mice, as well as in corresponding untreated mice, was visualised and
compared by SDS-PAGE followed by Western blotting (FIG. 3). The basal
levels of Cyp2b10 and Cyp3a11 in huPXR, huCAR and huPXR/huCAR mice are
compared to the basal levels observed in wild-type mice in FIG. 3. This
relative quantification shows that basal Cyp2b10 levels increase in the
order huPXR→huCAR→huPXR/huCAR. However, basal Cyp3a11 were
less markedly increased in huCAR mice. Cyp3a11 levels were increased to
an approximately equal extent (more than 2 fold) in both huPXR and
double-humanised huPXR/huCAR mice.

[0143] Treatment with the human-specific inducer rifampicin led to an
increase in the levels of Cyp3a11 in all mice. Whereas the administration
of rifampicin and phenobarbital in combination appeared to have no
additional effect in the wild type, induction of Cyp3a11 was somewhat
stronger in huPXR.

[0144] In this experiment, the activity of these transcription factors in
combination was determined by measuring the barbiturate induced sleeping
time. Sleeping time has been known for many years to be directly
proportional to the hepatic cytochrome P450 activity and this activity
can be at least in part ascribed to the P450 levels in the liver
determined by CAR and PXR function.

[0145] Mice were given a single intraperitoneal dose of Narcoren (sodium
pentobarbitone; purchased via a Veterinary Consultant; distributed by
Merial GmbH, Germany) at 25 mg/kg of body weight. The time taken for the
mice to lose, and subsequently to regain, their righting reflex was
measured. Results are given in Table 1 below:

[0146] Whereas wild type mice given a narcotic dose of pentobarbitone
slept for 21 minutes, the double humanised mice for CAR and PXR slept for
34 minutes. These mice therefore demonstrate a significant difference to
their wild type controls indicating that the double humanised mouse has a
marked difference in its response to drugs relative to the wild type
animals.

Summary of Work in Examples 1 and 2 Above

[0147] A model has been developed where human PXR is expressed in both the
liver and GI tract of mice in the predicted fashion at levels equivalent
to those of the endogenous gene. The PXR protein has been shown to be
functional as the mice are responsive to compounds known to induce gene
expression via this pathway.

[0148] Equivalent humanisation has also been achieved with respect to the
CAR gene (huCAR mice). Strain differences between wild type and the
humanised mice have been demonstrated and the humanised mice have been
shown to be more responsive to compounds known to be more active in
humans than in mice, i.e., to human PXR or human CAR rather than murine
PXR or murine CAR. The construction of knock-out lines has also been
confirmed for both the PXR and the CAR genes (koPXR and koCAR).

[0149] Moreover, mice which contained humanised PXR have also been crossed
into mice which contained humanised CAR to produce mice containing both
humanised PXR and humanised CAR.

Example 3

huPPARα and koPPARα

[0150] A DNA sequence encoding human PPARα has been inserted into
the mouse PPARα locus, as shown in FIG. 4, enabling expression of
human PPARα under the control of the mouse PPARα promoter.
The DNA sequence encoding human PPARα comprises at least part of
intron 5 and intron 6 of the human PPARα gene (FIG. 4). The
targeting vector(s) include sequence elements that enable Cre-mediated
PPARα knock-out to produce koPPARα (FIG. 4).

Example 4

Characterisation of huPPARα and koPPARα Humanised Mice

[0151] Six male C57BL/6J mice were obtained from Harlan, (UK). Three male
homozygous hPPARα mice were generated according to the protocols
herein by TaconicArtemis, Germany. All mice were sexually mature. On
arrival in the MSRU the mice were housed on sawdust in solid-bottom,
polypropylene cages. No environment enhancing materials was used during
treatment.

[0152] In the animal room the environment was controlled to provide
conditions suitable for the C57BL/6J and transgenic strains of mouse. The
temperature was maintained within a range of 19-23° C. and
relative humidity within a range of 40-70%. There was a nominal 14-15 air
changes per hour. Twelve-hour periods of light were cycled with
twelve-hour periods of darkness. For this study no special arrangement of
cages was used. The mice were allowed to acclimatise for a minimum of
five days following arrival in the test facility.

[0153] RM1 pelleted diet (supplied by Special Diet Services Ltd.,
Stepfield, Witham, Essex, UK) was used. The specification of the diet is
held by the MSRU, Dundee. Drinking water was taken from the local supply
and provided in bottles. Pelleted diet and drinking water was provided ad
libitum prior to and throughout the study.

[0154] Body and Liver Weights

[0155] Animals were treated either with 4 daily doses of Wy-14,643/corn
oil (50 mg/kg, orally) or with corn oil alone, as shown in Table 2.
Approximately 24 hr after the last dose, all mice were sacrificed using
an increasing concentration of CO2. Liver and plasma were collected for
analysis.

[0156] Following treatment with Wy-14,643 or the vehicle (corn oil), the
mice were sacrificed and their livers removed and weighed. Body weights,
liver weights and liver/body weight ratios of all mice were calculated
(Tables 3-5).

[0157] Dosing solution was prepared on the day of administration by adding
corn oil to the requisite quantity of inducing agent and stirring to
obtain a fine suspension. The concentration of inducing agent was of
supplied chemical, without any correction for purity. Animals were
administered vehicle or inducing agent, orally, as indicated in Table 2.
The volume administered was 10 ml/kg bodyweight. This route of
administration was chosen for consistency with previously published work.

[0158] On the day of termination the mice were weighed, the body weights
recorded, and then transferred to a suitable room for post mortem.
Approximately 24 hrs after treatment, the mice will be killed by exposure
to a rising concentration of CO2.

[0159] Absolute and relative liver weights were similar between
vehicle-treated WT and hPPARα mice and significant increases were
detected in both strains following treatment with Wy-14,643. Absolute
liver weights were increased following treatment with Wy-14,643 in WT
mice by ˜39% and in the two transgenic mice by 20 and 29%, when
compared with vehicle-treated mice.

[0160] Plasma Clinical Chemistry

[0161] The plasma was processed by taking blood from the terminated mice
by cardiac puncture into lithium/heparin-coated tubes. Following removal
into suitable tubes for plasma preparation, terminal blood samples taken
by cardiac puncture were mixed on a roller for 10 min then cooled on ice.
Red blood cells were removed by centrifugation (2,000-3,000 rpm for 10
min at 8-10° C.). Immediately after centrifugation, the
supernatant (plasma) was collected in eppendorfs and kept on wet ice.
Plasma was transferred into cryovials for clinical chemistry and
immediately flash frozen in liquid nitrogen, then stored at approximately
-70° C.

[0162] All clinical chemistry analytes measured in mouse plasma using the
COBAS Integra 400+ (Roche) had been optimized for human plasma samples
according to manufacturers' instructions. An extensive historical
database of clinical chemistry analytes had been generated and was used
to verify mouse samples assayed using the COBAS Integra 400+.

[0164] Concerning plasma markers of hepatotoxicity, ALT, AST and ALP
concentration appeared to be unaltered in both mouse lines following
treatment with Wy-14,643; however due to variability within the small
treatment groups no clear conclusions could be made concerning
Wy-14,643-mediated hepatotoxicity.

[0165] Plasma albumin levels were unaltered in both mouse lines following
treatment with Wy-14,643. On the limited number of mice available to
analyse, detectable bilirubin concentrations were variable between
animals and treatment groups. Plasma LDL, HDL and cholesterol levels were
unchanged in hPPARα mice when compared with WT mice, irrespective
of treatment. However, following treatment with Wy-14,643, triglyceride
concentrations were significantly decreased by ˜50% (p<0.05) in
WT mice and by 25 and 29% in the two hPPARα mice, compared to the
levels seen in the corresponding control mice. Interestingly,
triglyceride levels were 80% greater in the single vehicle-treated
transgenic mouse compared to those in vehicle-treated WT mice (n=3).

[0167] The liver was processed as follows. The gall bladder was removed,
and then the t liver was removed and weighed. Three pieces of liver (5
mm3) were removed and placed in separate cryovials, then flash
frozen in liquid nitrogen at approximately -70° C. for Taqman®
analysis and DNA sequencing.

[0168] Two samples of liver, approximately 2 mm strips, were taken, one
from the Left lobe and one from the Median lobe. These were placed in the
same scintillation vial containing 20 ml of 10% neutral buffered formalin
(NBF) for histology analysis.

[0169] The liver was weighed again, and then placed into ice cold 1.15%
(w/v) KCl prior to homogenisation and subcellular fractionation.

[0170] Fresh weighed livers were processed, according to a modified
version of CXR Laboratory Method Sheet (LMS) Cent-001, to homogenate,
nuclear, mitochondrial (heavy pellet), cytosolic and microsomal
fractions. The method was modified to allow for the collection of nuclear
fractions. Fresh liver samples were processed to 10% (v/v) homogenates as
according to Cent-001. Homogenates were placed 15 ml Falcon tubes and
centrifuged at 50 g for 5 mins at 4° C. to remove cell debris. The
remaining supernatant was transferred into 10 ml centrifuge tubes and the
pellet was discarded. The supernatant was topped up to 10 ml with ice
cold SET buffer then centrifuged at 700 g for 10 mins at 4° C. The
resulting supernatant was retained for further fractionation to
mitochondrial, cytosolic and microsomal fractions.

[0171] The pellet (unwashed crude nuclear membranes) was resuspended in 10
ml ice-cold SET buffer, homogenised with 2-3 passes and then centrifuged
at 700 g for 10 mins at 4° C. The resultant supernatant was
discarded and the pellet (washed crude nuclear membranes) was resuspended
in 1 mL/g original tissue ice-cold SET buffer and homogenised by 2
passes. All fractions were stored at approximately -70° C. prior
to analysis.

[0172] Following fixation, both liver sections from each animal were
processed then paraffin embedded. Wax blocks will be stored at room
temperature

[0173] Immunoblot Analysis of Cyp4a Protein Expression

[0174] To investigate the downstream effects of PPARα activation,
the expression of Cyp4a proteins was evaluated by immunoblot analysis in
liver microsomes from WT and hPPARα mice.

[0175] Individual liver microsomal samples were analysed by immunoblotting
for Cyp4a. Protein concentration was measured using the CXR microlowry
method. Quantification of Cyp4a protein in mouse liver microsomes was
carried out. A murine his-tagged Cyp4a recombinant standard was loaded
onto the NU-PAGE gel. The results are shown in FIG. 17. The presence of a
signal at approximately 50 kD in a positive control sample (ammonium
perfluorooctanoate (APFO)-induced rat liver microsomes) demonstrated that
the antibody used recognised a protein of the correct molecular weight.

[0176] An immunoreactive band specific to Cyp4a was detectable at 50 kD in
both vehicle-treated WT and hPPARα mice. Following treatment with
Wy-14,643, marked induction was observed in WT mice, consistent with
upregulation of Cyp4a protein expression. A similar effect was seen in
the hPPARα mice. This is consistent with the hypothesis that human
and murine PPARαs have similar affinities for Wy-14,643.

[0178] The effect of Wy-14,643 administration on cyanide-insensitive
palmitoyl-CoA oxidation was investigated for the heavy pellet fraction
(crude mitochondria) from each available mouse liver as a marker of
peroxisomal enzyme activity, known to be regulated by PPARα. This
assay was used to confirm the presence of a functional PPARα gene
in WT and hPPARα mice. Wy-14,643 induced cyanide-insensitive pCoA
oxidation in crude mitochondrial fractions from both mouse lines,
exhibiting approximately 3-fold induction relative to vehicle controls
(FIG. 18, Table 16).

[0180] Liver microsomes from each available animal were evaluated for
Cyp4a activity by measuring lauric acid hydroxylation (FIG. 19, Tables
17-18). Similar increases in formation of both metabolites (12-hydroxy;
12-OH and 11-hydroxy; 11-OH) were observed in both lines following
treatment with Wy-14,64{tilde over (3)}. Interestingly, 12-OH lauric acid
hydroxylation activity was lower in the vehicle-treated hPPARα
mouse when compared to those seen in WT mice.

[0182] RT-PCR was performed on liver samples from a single vehicle-treated
WT mouse and a single hPPARα mouse) to assess whether the
full-length human PPARα transcript was present in the hPPARα
mouse.

[0183] Total RNA was prepared from one control WT and one hPPARα
mouse liver tissue samples, and the RNA samples were purified using
RNeasy kit (QIAGEN, Cat No. 74104). RT-PCR was conducted by using
Superscript III One-Step RT-PCR Platinum Taq HiFi Kit (Invitrogen Corp.
Cat. No. 12574-030), following the manufacturer's protocol. The products
of RT-PCR were separated by electrophoresis on an agarose gel.

[0184] In order to confirm that the human PPARα transcript had a
correct translation start site, primers termed PPARα-F and
PPARα-R were used in the RT-PCR, as shown in FIG. 20. The expected
product of RT-PCR, having a molecular weight of 1.4 kb, was detected
(FIG. 21). The resulting cDNA was cloned and characterised by sequence
analysis.

[0185] The targeting vector was designed in such a way that a chimeric
construct of genomic and cDNA consisting of Exons 3-5, Intron 5, Exon 6,
Intron 6 and Exon 7-8 of human PPARα was introduced into the mouse
genome, replacing the coding region of Exon 3 of the murine PPARα
gene. The construct was designed so that the transcript would be
terminated by a polyA motif. The targeting vector carries an FRT-flanked
neomycin resistance cassette, inserted into human Intron 5, which should
be removed by FLP-mediated recombination to generate a humanised
PPARα allele. Therefore, primers were designed to anneal to the
construct's polyA region. The primers were as follows:

[0186] The specificity of the primers used was confirmed by the absence of
a detectable band at the correct molecular weight indicating that, as
expected, no human PPARα mRNA was present in the WT mouse analysed
(FIG. 21). RT-PCR data demonstrated that at least two products (1.2 kb
and 1.4 kb) could be generated from hPPARα mouse liver RNA, but not
from WT mouse RNA. Although the expression of both fragments was low,
subsequent cloning and sequencing analysis demonstrated three distinct
transcripts, as described below.

[0187] RT-PCR was conducted using the Superscript III One-step RT-PCR
Platinum Taq High Fidelity Kit (Invitrogen Corp, Cat #10574-030)
according to the manufacturer's protocol. Total RNA (1 μg) was
prepared from a vehicle-treated hPPARα mouse and mouse a
vehicle-treated WT mouse and used directly for RT-PCR using primer pair
PPARα-F and PPARα-R.

[0188] Duplicate 50 μl synthesis reactions were set up for each and run
under the following conditions;

[0189] cDNA synthesis: 1 cycle of;

[0190] 54° C. 30 min

[0191] 94° C. 2 min

[0192] PCR amplification: 40 cycles of;

[0193] 94° C. 20 sec

[0194] 58° C. 30 sec

[0195] 68° C. 2 min

[0196] Final extension: 1 cycle of;

[0197] 68° C. 5 min

[0198] Restriction Analysis of Clones

[0199] A variant human PPARα mRNA species has been identified
(Gervois et al, 1999). Sequence analysis revealed that the variant
contained a 203 bp deletion and that the deleted fragment localized
exactly at the boundaries of Exon 6, indicating that it is generated by
an alternative splicing event skipping Exon 6. This resulted in a frame
shift introducing a premature stop codon. The shorter transcript was
predicted to result in the production of a truncated hPPARα protein
lacking part of the hinge region and the entire ligand-binding domain.
RNase protection analysis demonstrated that variant human PPARα
mRNA was expressed in several human tissues and cells, representing
between 20-50% of total PPARα mRNA. By contrast, variant
PPARα could not be detected in rodent tissues. Thus, the
PPARα variant transcript appeared to be specifically expressed in
man.

[0200] In order to confirm that the 1.2 kb product observed following
RT-PCR of hPPARα mouse liver RNA is the reported alternative
spliced variant of human PPARα, the products of RT-PCR were
separated by agarose gel electrophoresis using Qiagen gel purification
kits. Fragments in the range of 1.2 to 1.4 kb were extracted from the
gel, purified, ligated into the T/A site of the pCR4-TOPO vector using
TOPO TA Cloning kit for Sequencing (Invitrogen Corp. Cat. no. K4575-01)
and transformed into TOP10 ultracompetent cells. Colonies were screened
by digestion with restriction enzyme BglII to determine the presence of
an insert within the vector. One clone (clone #1) containing a 1.2 kb
insert and six clones (clone #2, 6, 7, 9, 24 and 36) containing a 1.4 kb
insert were sequenced. The ligated DNA was transformed into DH5α
ultracompetent cells (Advantage, Dundee) and plated onto ampicillin
plates to select for positive clones.

[0201] Sequence Analysis of Clones

[0202] Plasmid DNA for each positive clone was prepared using the Qiaprep
Spin Miniprep kit according to the manufacturer's protocol (Qiagen; Cat
#27160) and eluted in 50 μl elution buffer.

[0203] Approximately 300 ng of plasmid DNA was digested with BglII to
release the insert from the vector backbones. The digests were loaded
onto 1% agarose TAE gels.

[0204] Colonies resulting from the transformation plates were picked and
added directly to a 20 μl PCR mix,

[0216] Sequence comparisons confirmed that the 1.2 kb insert in clone #1
was the spliced variant of human PPARα which lacked Exon 6 (SV1, as
shown in FIG. 22). The 1.4 kb insert represents two types of transcripts,
the normally spliced version (clone #2, 7, 9, and 24) and a splice
variant SV2. Variant SV2 is a previously undescribed transcript using an
alternative intronic 3' splice site resulting in the addition of a 4 bp
(GTAG) out-of-frame insertion into the 3' end of Exon 5 and generation of
a premature stop codon (FIG. 22). Based on the agarose gel
electrophoresis, the ratio of 1.4 kb transcript to 1.2 kb transcript
appeared to be about 1:1. However, the ratio of normally spliced
transcripts to alternative spliced variants SV1 and SV2 could not be
determined using the RT-PCR method.

[0217] The alternatively spliced variants SV1 and SV2 encode truncated
PPARα proteins containing 174 and 180 amino acids, respectively.
Both truncated proteins lack a large region of the ligand binding domain
(LBD), but they still contain the DNA binding domain (DBD) (FIG. 23). It
remains to be determined whether the two alternative spliced transcripts
can be transformed in hPPARα mice to form the truncated protein and
whether they can bind to PPRE-containing DNA fragments. It has been
reported that although truncated PPARα (PPARαtr) could not
bind to PPRE elements in gel retardation assays, nuclear hPPARαtr
was a potent repressor and could affect the transcriptional activity of
full length hPPARα protein in vitro (Gervois et al, 1999).

[0218] In summary, a full-length human PPARα transcript specific to
the humanised mouse was detected, cloned and verified by sequencing. Two
alternatively spliced variants (SV1 and SV2) were identified in this
study. Variant SV1 has been published and is a human specific
alternatively spliced variant (Gervois et al, 1999). Variant SV2 is a new
type of transcript with the addition of a 4 bp (GTAG) out-of-frame insert
at the 3' end of exon 5, resulting in a premature stop codon. The
potential functions of truncated PPARα protein in humanised mice
are not known.

[0221] Primers specific for murine PPARα and human PPARα
(Assay-on-demand Cat #, Mm00440939_m1 and Hs00947539_m1, Applied
Biosystems, respectively) were employed for TaqMan® analysis of
PPARα expression in mouse liver from WT and hPPARα mice. The
murine PPARα primers were designed to anneal between Exons 7 and 8
of the murine PPARα sequence and the human PPARα primers were
designed to amplify Exons 7 to 9 of the human sequence.

[0222] Levels of murine and human PPARα mRNA were quantified by
quantative RT-PCR (TaqMan®) in the site of normal PPARα
expression (liver) in WT and hPPARα mice (Table 19). RNA was
extracted from mouse liver. cDNA was synthesised from all available RNA
samples, and TaqMan® analysis was performed in all available samples
using primers specific of murine PPARα and human PPARα
(Assay-on-demand kit Applied Biosystems). Murine α-actin was used
as the internal standard (Assay-on-demand kit Cat #43S2933E, Applied
Biosystems).

[0223] Human PPARα transcripts were found in hPPARα mice, but
not in WT mice. Murine PPARα was identified in WT mice but not in
the hPPARα mice. The level of PPARα mRNA detected was similar
in both models.

TABLE-US-00020
TABLE 19
TaqMan® analysis of PPARα mRNA in mouse liver. Total RNA was
isolated
from livers of WT and hPPARα mice. Subsequent cDNA synthesis and
qRT-PCR was
performed with specific primers for murine or human PPARα. Values
represent cycling
time (CT) values for individual mice. The internal standard used for each
assay was
murine α-actin. Grey boxes denote CT values <35 = no mRNA
expression. The lower
the CT value, the greater the level of mRNA expression.
##STR00001##

[0224] Mice have now been generated that are double homozygous for human
CAR and PPARα and heterozygous for human PXR. FIG. 25 shows PCR
confirmation of these double homozygous CAR/PPARα and heterozygous
PXR humanised mice. Mice with the IDs m 245218, f 245221, f 245226 and f
245227 are homozygous humanised for CAR and heterozygous humanised for
PXR. Triple humanised PXR/CAR/PPARα mice will be available very
shortly, following protocols set up in existing breeding programs at
TaconicArtemis.

[0226] Mice that are triple homozygous humanised for PXR, CAR and AHR have
now been generated. FIG. 24 shows PCR confirmation of triple homozygous
PXR/CAR/AhR humanised mice. Mice with the IDs f 241998, f 242001 and f
242004 are triple homozygous humanised for PXR, CAR and AHR. Such mice
will of extreme value in the assays described herein.

Example 6

Proof of Concept: the Effect of the Non-Genotoxic Carcinogen, PB, in
Rodents

[0227] Phenobarbital (hereafter PB) is known to cause liver cancer in mice
and rats but does not do the same in humans. Following long term
treatment to PB, rodents develop liver tumours. Initially, PB causes a
hyperplastic response and cell replication and liver weight increases for
the first two weeks of treatment. However, after approximately two years,
liver tumours become evident in treated animals. Under this type of
analysis, PB would be deemed unsafe for use in humans. However, PB is
indeed safe, having been sold for many years with no record of liver
tumour incidence in treated patients. This illustrates the shortcomings
of current animal models to test for drug safety in humans, and means
that there is unnecessary drug attrition occurring at this stage of the
safety testing process. The question which drug companies need to answer,
at as early a stage of testing as possible, is whether hyperplastic
responses to chemicals observed in animals are actually relevant to man?

[0228] The transcription factor CAR is known to be essential for responses
to PB-like inducers. Wei et al, 2000 (Nature) showed that in wild type
mice, CAR activators increased liver mass, reflective of cellular
hypertrophy and hyperplastic response. In contrast, CAR KO mice showed no
increased liver mass after treatment with CAR activators. Furthermore,
induction of DNA synthesis as determined by increased incorporation of
BrdU in wild type mice was also absent in CAR KO mice. Similarly, Cheung
et al, 2004 showed that humanisation of the mice for PPARα
decreased the increase in liver weight elicited by treatment with various
drugs in wild type animals. The humanised mice also showed a lack of
increased replicative DNA synthesis, as seen in the wild type animals.

[0229] In order to assess whether humanised PXR/CAR mice mimic the
response to PB in humans, the huPXR/huCAR and PXRKO/CARKO mouse models
were used. The mutant mouse strains were obtained from Artemis. The WT
mouse strain C57BL/6J was obtained from Harlan (UK). All animals were
between 10 and 16 weeks of age.The following parameters were studied:
[0230] Liver/body weight ratios [0231] BrdU incorporation analysed as a
measure of cell proliferation [0232] Haematoxylin and eosin (H&E) liver
histopathology [0233] Expression and activity of P450s by SDS-PAGE and
Western blotting in liver microsomes [0234] Apoptotic indices as analysed
by TUNEL assay in the liver

[0235] The study consisted of 6 groups with 10 WT mice per group, 9-10
PXRKO/CARKO mice per group and 9 huPXR/huCAR mice per group (Table 19).
All animals were implanted with osmotic pumps (Alzet 2001) containing
bromodeoxyuridine (BrdU, 15 mg/ml in phosphate buffered saline [PBS],
pH7.4) 5 days before termination for all mice (Day -1). Post operation
all animals had no abnormalities detected. On Day 1 all animals were
dosed with either 80 mg/kg PB/saline or saline alone by intraperitoneal
injection for 4 days as detailed in Table 19.

[0237] Following treatment with PB or the vehicle, the mice were
sacrificed and their livers were removed and weighed. Hepatomegaly was
observed in both the WT and "humanised" mice, but not in the PXRKO/CARKO
in response to PB treatment, as shown by increases in liver body weight
ratios of 118% and 122%, respectively (Table 20, FIG. 7).

[0239] All mouse liver and duodenum sections were analysed for BrdU
incorporation as a measure of cell proliferation. The method used was an
indirect BrdU labelling assay. PB increased the hepatocellular labelling
index (S-phase) in the WT mice by approximately 5-fold and appeared to
have no effect on cell proliferation in the huPXR/huCAR or PXRKO/CARKO
(FIG. 8, Table 21).

[0240] Liver in situ Cell Death

[0241] A 50% inhibition of hepatocellular apoptosis by non-genotoxic
carcinogens has been previously demonstrated with consistency in rats.
However, no such consistency has been observed in mice. An indirect TUNEL
labelling assay was used to analyse hepatic in situ death (FIG. 9/Table
21). The present study has shown a marker variation in apoptotic indices
in mouse liver. Thus, small (e.g. 50%) compound-induced decreases, upon a
low background, were not readily demonstrable.

[0242] H&E Analysis

[0243] Two samples of the liver (one from the lobe, one from the median
lobe) and one sample of the small intestine were taken and preserved in
4% neutral buffered formaldehyde (NBF). The preserved liver samples of
all mice of all groups were trimmed, processed and embedded in paraffin.
The paraffin-embedded samples were sent to Progenix, Inverkeithing, UK
where they were sectioned at a nominal thickness of about 5 {acute over
(.ae butted.)}m and then stained with haematoxylin and eosin (H&E). One
section of each organ sample was examined by Dr. Ortwin Vogel, Consultant
Pathologist, Kiel, Germany. Subsequent to his histopatholoical analysis
of all H&E stained mouse livers and small intestines, Dr. Vogel reported
the following finding;

[0244] Microscopically, a slight to moderate centrilobular hepatocellular
hyperthrophy was noted, in PB treated huPXR/huCAR and WT animals (FIG.
10). This finding is considered to be related to the treatment with PB.
In contrast, no unequivocal evidence of a hepatocellular hypertrophy was
noted in PXRKO/CARKO mice following treatment with PB. In addition,
mitotic figures indicating hepatocellular proliferation were noted mainly
in PB-treated WT mice but also, with a lower incidence, in the
"humanised" and null mouse lines.

[0245] All other microscopic findings recorded in the liver did not
significantly distinguish PB-treated mice from control mice or the
differences were regarded as random events. All these findings are
considered to be spontaneous in nature and within the normal background
pathology commonly seen in mice. No microscopic findings were recorded in
the small intestine. Under the conditions of this study, PB (80 mg/kg/4
days/IP) produced pathological evidence of a hepatocellular
hypertrophy/hyperplasia in mice of the WT strain as well as in mice of
the huPXR/huCAR strain. Evidence of hepatocellular hyperplasia occurred
in PB-treated mice of the PXRKO/CARKO strain.

[0246] Hepatic P450 Induction

[0247] P450 catalytic activities in the liver microsomal fractions were
quantified. For the quantification of mouse Cyp2b10 and Cyp3a11
activities, the dealkylation of pentoxyresofurin (PROD) and the
debenzylation of benzyloxyquinoline (BQ) activities, respectively. In
addition, benzyloxyresorufin-O-demethylase (BROD),
methoxyresorufin-O-demethylase (MROD) and 7-ethoxyresorufin-)-deethylase
(EROD) activities were also measured and evaluated (see FIG. 11).

[0248] EROD activity is indicative of Cyp1a1/1a2 and Cyp1b1 in the mouse,
whereas MROD is a substrate for Cyp1a2. However, other isoforms may be
contributing to the measured activities. The results from the two enzyme
assays are in reasonably good agreement with each other (FIG. 11a-b) As
the Cyp1a2 gene is constitutively expressed, this may explain the basal
levels of activity observed in both activity assays. Cyp1a1 is only
expressed following induction in mice (Ikeya et al, 1989) and
Phenobarbital has been reported not to induce this P450 in C57BL/6J mice
(Sakuma et al, 1999). The MROD and EROD data demonstrated that enzyme
activities were increased by PB treatment in WT and huPXR/huCAR mice.
Both assays revealed no increase due to PB treatment in the PXRKO/CARKO
line. Earlier data using the PXRICAR mouse panel demonstrated PB, at 40
mg/kg/4 days, could activate Cyo1a2 via the CAR mediated pathway,
although other data are consistent with these earlier findings.

[0249] A 5-fold increase in BQ activity was seen in the PB-treated
huPXR/huCAR mice, whereas a marginal increase was detected in the WT
animals (FIG. 11c). These data demonstrate a clear species difference
between the mouse lines, indicating that the human receptors have a
greater sensitivity to PB than their murine counterparts.

[0250] Furthermore, this mechanism appears to be dependent on the presence
of the receptors, as verified by the absence of Cyp3a11 induction in the
PB-treated PXRKO/CARKO mice.

[0251] BROD and PROD are markers of Cyp2b10 activity in the mouse. In both
the WT and huPXR/huCAR mouse lines, marker Cyp2b10 induction was
observed, at similar levels following treatment with PB (FIG. 11d-e).
However, PROD or BROD activities were not altered in the PXRKO/CARKO mice
upon exposure to PB. Overall, PB induced P450 catalytic activities in the
WT and huPXR/huCAR mouse lines, but not in the animals which were devoid
of these receptors. This clearly indicates that PB-mediated P450
induction is CAR/PXR-dependent.

[0252] In accordance with P450 activity data, quantification of Cyp2b10
and Cyp3a11 protein in pooled mouse liver microsomes by Western blotting
revealed that both P450s were induced by PB in WT and humanised mouse
lines but not in the PXRKO/CARKO animals (FIG. 12). Furthermore, the
species difference in Cyp3a11 induction is confirmed at the protein
levels, suggesting that PB has a greater sensitivity for the human
receptors over its murine equivalents.

[0253] In conclusion it can be said that hepatomegaly occurred only in WT
mice and in huPXR/huCAR mice. The KO PXR/KO CAR showed no effect. The
same pattern was mirrored for P450 induction. However, the most striking
result was found when the mice were tested for hepatocellular
proliferation (as determined by incorporation of the DNA precursor BrdU),
where it was found that only the WT mice displayed a proliferation of
hepatocytes. Both KO and humanised animals showed no proliferative effect
whatsoever.

[0258] These mice and huPXR/huCAR/huPPARα will be of considerable
value in assessing the true hazard of non-genotoxic rodent "liver growth
carcinogens" to humans. They will also provide useful tools to unravel
the complexities of xenobiotic-induced liver growth and species
differences in such growth.